Dynamic assembly and disassembly of the human DNA polymerase δ holoenzyme on the genome in vivo, Cell Reports 2019
Description
Live Cell Single Molecule Tracking Data of Halo-PCNA and SNAP-PolD examining holoenzyme assembly pathways
Files
Steps to reproduce
Movies were collected on a custom 2D HILO single molecule microscope. Movies were then processed with slimFAST_2 for particle localization and tracking according to parameters listed in the README file. Data from slimFAST_2 was further processed using evalSPT to obtain a format suitable for further downstream processing. The output from evalSPT_3 is then used as input for Matlab script spt_process_v15CR.m file. The resulting data structure (sptana) contains metadata, nuclear segmented binding trajectories, and nuclear segmented dwell times for binding trajectories (ndwet). Each row is a separate cell. Sptana data structures should from 2 channel SMT should be organized to have subsequent rows containing cells from channel 1 data followed by subsequent rows of matched cells from channel 2. For example, 10 cells of 2 channel SMT should have rows 1-10 channel 1 information from Cells A-J followed by rows 11-20 channel 2 information from Cells A-J. The sptana data structure containing matched cells with 2 channels of tracking is used by the livecellColocV1CR.m file to isolate spatially and temporally colocalized molecules. The output This program saves the following values, percentages of molecules colocalized in each cell (perColoc), percentages of molecules temporally colocalized in each cell (perClcT),aggregated arrival/departure times of channel/color 2 molecules with respect to channel/color 1 (ADBag), aggregated colocalization times (ClcTag), and a cell structure containing a table with all molecular IDs, arrival departure frames, XY position, arrival/departure times of channel/color 2 with respect to channel/color 1 (FinalMoldata)). The sptana data structure is used by sptclst_v24CR.m file to generate maps of hubs(clusters) of binding events. The resulting data structure (sptclst) contains metadata, cluster numbers, locations and Molecular IDs of binding events in clusters. All software used for processing this data along with output formats can be found on https://github.com/Coleman-Laboratory/SMT-Data-and-code.